This application describes a research program designed to study the membrane components and mechanism of Methotrexate (MTX) transport (via the 5-methyltetrahydrofolate system) in L1210 cells. The results will contribute to an understanding of how MTX enters cells during chemotherapy. The MTX-binding activity of intact L1210 cells will be quantitated and characterized with regard to substrate specificity, pH-and ion-dependence, and response to inhibitors. The relationship between binding activity and the transport of MTX will be examined in mutant cell-lines that contain an altered ability to transport MTX. Other studies on the mechanism of MTX transport will include establishing the energy source for the concentrative uptake of MTX and the role of cyclic AMP in regulating this process. L1210 cells also contain a low-affinity, folate uptake system whose substrate specificity, ability to transport MTX, and possible regulation by cyclic nucleotides will be analyzed.